【摘要】：目的:建立一種快速便捷的亞硫酸氫鹽修飾DNA方法用于DNA甲基化檢測(cè)。方法:提高亞硫酸氫鹽濃度及修飾溫度、縮短修飾時(shí)間并用玻璃奶回收DNA改進(jìn)DNA修飾方法,用亞硫酸氫鹽測(cè)序法分析MAGE-A3基因和DAP-K基因目的片段中胞嘧啶轉(zhuǎn)化為胸腺嘧啶的效率,評(píng)價(jià)改進(jìn)方法與傳統(tǒng)方法、試劑盒方法對(duì)DNA的修飾效果。結(jié)果:改進(jìn)的方法可將操作過(guò)程縮短到3 h左右;非甲基化的胞嘧啶到胸腺嘧啶的轉(zhuǎn)化率超過(guò)99%,與傳統(tǒng)方法、試劑盒方法相比差異無(wú)統(tǒng)計(jì)學(xué)意義(χ~2=0.0564,P0.05);只針對(duì)非甲基化的胞嘧啶進(jìn)行轉(zhuǎn)化修飾,而在對(duì)甲基化胞嘧啶過(guò)度修飾為胸腺嘧啶方面與傳統(tǒng)方法無(wú)統(tǒng)計(jì)學(xué)差異(χ~2=0.0149,P0.05)。結(jié)論:改進(jìn)的亞硫酸氫鹽DNA修飾方法修飾效率高,對(duì)甲基化的胞嘧啶過(guò)度修飾影響不顯著,且具有節(jié)省時(shí)間,操作簡(jiǎn)便等優(yōu)點(diǎn)。
[Abstract]:Objective: to establish a rapid and convenient method for the detection of DNA methylation by hydrogen sulfite modified DNA. Methods: the concentration and temperature of hydrogen sulfite were increased, the modification time was shortened, and the DNA modification method was improved by recovering DNA from glass milk. The conversion efficiency of cytosine to thymine in the target fragment of MAGE-A3 gene and DAP-K gene was analyzed by bisulfite sequencing. The modified method, traditional method and kit method were evaluated for the modification effect of DNA. Results: the improved method could shorten the operation process to about 3 h, and the conversion rate of unmethylated cytosine to thymine was more than 99%, there was no significant difference compared with the traditional method and kit method (蠂 ~ 2 ~ 2, 0.0564g, P0.05). Only the unmethylated cytosine was modified by transformation, but there was no significant difference between the traditional method and the hypermethylated cytosine (蠂 ~ 2) 0.0149 (P0.05). Conclusion: the modified DNA modification method has the advantages of high modification efficiency, little effect on methylated cytosine overmodification, time saving, simple operation and so on.
【作者單位】： 南陽(yáng)理工學(xué)院張仲景國(guó)醫(yī)學(xué)院;南陽(yáng)市第一人民醫(yī)院神經(jīng)內(nèi)科;中山大學(xué)達(dá)安基因股份有限公司;
【分類號(hào)】：R440
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本文編號(hào)：2364358